Biologic drugs pose several unique manufacturing and regulatory challenges due to their intrinsic and complex profile. They have a high level of structural complexity and heterogeneity, are produced in living systems or supplemented with reagents derived from living systems, and consequently have a complex purity/impurity profile that poses unique analytical challenges, particularly from a quality and safety perspective.
Ensuring the safety of biologic drugs is important. Contamination, including the presence of pathogenic microorganisms, such as viruses, may cause serious illness or even death in certain patients to whom such drugs are administered. Indeed, regulatory approval of such drugs depends, in part, on eliminating, or at least reducing the levels of pathogenic microorganisms in the drug to acceptable levels. After removal of microorganisms from the biologic drugs, the drugs are screened to confirm the removal of the microorganisms.
In contrast to cellular microorganisms such as bacteria, which can be multiplied prior to their detection, most virus particles need to be directly detected in the respective sample. Generally, the presence of virus is detected by way of PCR-based amplification and detection of viral DNA or RNA. It is, thus, important to have sensitive detection methods available for screening and detection of viral contaminants. Current detection methods, while sensitive, nevertheless still suffer from the drawback of producing both false negative and false positive results. The former is problematic insofar as it may result in a patient receiving a contaminated drug, and the latter is problematic insofar as it may result in a good batch of drug being destroyed or subjected to further expensive processing.
Pancrelipase is a biologic drug. This drug is a porcine-derived mixture of digestive enzymes, including pancreatic amylase, pancreatic lipase, and pancreatic proteases. It is used to enable patients with pancreatic insufficiency to better digest their food. Pancrelipase is prepared from porcine pancreases and, comprises, in addition to the amylase, lipase, and proteases, various types of other enzymes, salts, nucleic acids and carbohydrates. Pancrelipase is highly enzymatically active, having been prepared for this specific property, and in this respect, it is unlike other biological matrices and biological drugs. Being prepared from a biological source, the precursor of the pancrelipase end product may contain human pathogens such as viruses, which must be removed.
The methods used to extract the digestive enzymes from the pancreas glands, to form pancrelipase, result in a reduction in viral number and in viral infectivity through destruction of the viral envelope or capsid. Some viruses are able to survive the processing steps, although infectivity of such viruses may be degraded. Examples of such viruses include the Hepatitis E Virus (HEV).
To establish that pancrelipase is HEV-free, samples are subject to PCR-based amplification and detection of HEV RNA. The inference of the presence of virus, and therefore viral contamination of the pancrelipase, is made through the detection of base sequences specific to the virus. Viral RNA may be present in free form (absent any viral capsid) as an artifact of the pancrelipase production processes, or may be present encapsidated or intact as contaminating viruses. The former is not generally grounds for concern, although the latter indicates viral contamination. Unfortunately, however, current amplification and detection methods do not distinguish between free and encapsidated or intact viral RNA. Pancrelipase is a very difficult product on which to perform analyses of biological constituents, due to the enzymes thereof interfering with analyses, for example by the enzymes reacting/converting biological constituents of the sample to be tested or components used to effect the analysis. Therefore, detection methods and analytical techniques need to be specifically tailored for use with pancrelipase samples.
Before encapsidated RNA can be detected, it must be released from the envelope or capsid. Viral nucleic acids are released by treating tissue or other samples with a lysis buffer which destroys the protein viral capsid. The nucleic acid is then purified and amplified using PCR, and sequences specific to the virus of interest are detected through single strand nucleic acid probes containing a fluorphore.
Failure to distinguish between free and encapsidated intact viral nucleic acids may generate a false positive result for the presence of the virus in the sample. Preparation of pancrelipase may cause encapsidated viral RNA to be released during the extraction process. The lysis buffer results in the release of the encapsidated RNA into the general environment making it indistinguishable from free RNA. In some cases, the free RNA may constitute short fragments of the original nucleic acid strand.
Because current HEV RNA detection methods do not differentiate between free form RNA or fragments thereof, and encapsidated RNA in pancrelipase, improved detections methods are in need, particularly detection methods that can effectively differentiate between free form and encapsidated RNA, thereby reducing false positive test results.
In a publication by Schielke et al., Virology Journal 2011, 8:487, the investigators evaluated the presence of HEV in a boar liver homogenate. The methodology added ribonuclease (RNase) to the homogenate to degrade all RNA not protected by the viral capsid, prior to RNA amplification. The investigators observed that free RNA was purged from the homogenate by way of the RNase such that subsequent analyses detected only RNA from whole HEV virions (following lysis of the capsid to release the encapsidated RNA). The methods described in this publication are particular to liver homogenate. Persons having ordinary skill in the art would readily understand that enzymes in the pancreas, particularly pancreatic proteases, readily degrade nucleases such that RNase used in a liver extract would not necessarily function in a pancreatic extract.
Accordingly, there exists a need in the art for a method of detection that is able to differentiate between free and encapsidated nucleic acid to avoid the generation of false positive results in biological samples of the pancreas. There also exists a need in the art for a product of a sample that is free from free nucleic acid to detect an intact virus in biological samples of the pancreas.